1. Field of the Invention
This invention relates to the production of virus-resistant transgenic animals.
2. Information Disclosure Statement
Viral infections in agricultural animals represent a major economic loss for farmers and ranchers. Significant viral diseases in cattle are infectious bovine rhinotracheitis, parainfluenza, bovine viral diarrhea, rabies, and foot and mouth disease. In swine, transmissible gastroenteritis, swine mycoplasmal pneumonia, pseudorabies, and rabies infections are serious health problems. Poultry production is hampered by Marek's disease, Newcastle disease, infectious bursal disease, and infectious bronchitis, all of which are viral diseases. In 1981, over $500,000,000 was spent to treat agricultural animals against viral infections and the economic loss to agriculture resulting from viral disease is substantially greater that this cost.
One method of providing virus resistance is by use of vaccines. Vaccination may be with attenuated or inactivated virus, or with viral proteins produced by genetic engineering techniques. Vaccination, unfortunately, has many disadvantages. The animals must receive separate immunizations for each disease-causing organism. Moreover, the immunity diminishes with time and must be restored by follow-up immunizations, a practice which is inconvenient in the case of livestock allowed to roam on free range.
Another approach is that of treatment with an antiviral drug. Such drugs include acyclovir, ribovirin, adenine arabinoside and amantadine. A drug of particular interest is interferon, or, more especially, the various species of alpha (leukocyte) and beta (fibroblast) interferon. Collectively, these are called Type I interferons. The antiviral activity of various mammalian interferons is reviewed in Colowick, S. P. and Kaplan, N. O. Interferons, S. Pestka (Ed.), Methods in Enzymology. Orlando: Academic Press, Inc., 1986, Vol. 119(C). The interferons do not act directly against the virus, rather, they stimulate the antiviral response of the immune system. The antiviral activities of exogenous interferons in animals are reviewed by Stewart II, THE INTERFERON SYSTEM, 282-283, Table 16 (2d ed. 1979). Unfortunately, the antiviral drugs are expensive and provide protection for only a short period of time.
Kawade, et al., at the 1986 ISIR-TNO Annual Meeting, September 7-12, 1986, reported the production of transgenic mice carrying mouse interferon beta or gamma genes coupled to the mouse MT-I or heat shock promoters. According to Kawade, linearized plasmid DNA was microinjected into fertilized mouse eggs. Twenty-eight pups were born, four of whom carried the chimeric IFN genes. Three of these transmitted the genes to offspring. Apparently, Kawade had not determined whether the chimeric gene was actually expressed in the mice, but had already been able to express it in cultured cells induced by Cd.sup.2+ or by heat. Our invention relates to the introduction of the interferon gene of another animal species into a host animal, and to the production of a healthy transgenic animal with enhanced viral resistance which it can genetically transmit to its offspring.
Gordon and Ruddle, Science, 214:1244 (1981) claimed integration and stable germline transmission of genes injected into mouse pronuclei. Ten thousand copies of a human leukocyte IFN gene were microinjected into each zygote. Ten mice were born from 33 implanted embryos; one of the mice was transgenic. Expression was not reported, and the purpose of the study was to show that the technique could be used to study gene action during mammalian development.
Staehelin, WO 87/00864 (publ. February 12, 1987) observed inhibition of influenza virus in NIH 3T3 mouse cells genetically modified to express the mouse protein Mx. This protein is one whose production is stimulated by mouse interferon. Staehelin suggested that an animal could be protected against viral infection by inserting a gene encoding the Mx protein into that animal, preferably at the single cell embryo stage. He taught that the gene of choice would be the one encoding the Mx protein that is normally found in the species of animal to be protected. He did not suggest use of the interferon gene, or of a gene derived from a different species than the host.
The expression of an exogenous gene in a transgenic animal is a far more subtle process than the expression of the same gene in mammalian cell culture. Expression of the gene at the wrong time may kill the animal. The problem is particularly acute when the gene is inserted into an embryonic cell, since the developing embryo, fetus or neonate may be distinctly sensitive to the gene product. It is known that mouse interferon is toxic to newborn mice. Gresser, et al., Nature, 258:76-78 (1975).
There is considerable controversy as to the cross-species antiviral activity of interferon.
Wilkinson and Morris, Methods Enzymol., 119:96 (1986) reported that human IFN-beta had little antiviral activity on bovine EBTr (0.2) and murine L-929 (0.03) cells (activity on human GM 2767 cells taken as 100). McCullagh, et al., J. Interf. Res., 3:97 (1983) credits human beta-IFN with somewhat greater activity on mouse cells, but still much less than on human cells.
With respect to IFN-alpha, species, A, B, C, D and F all showed low antiviral (VSV) activities (0.5-5) on mouse L-929 cells relative to WISH cells (100), according to Weck, J. Gen. Virol., 57:233 (1981). Streuli, et al., PNAS, 78:2848 (1981) credited alpha-1 interferon with a somewhat higher activity on L929 cell (10% of activity on bovine cells, activity in WISH cells was 20% of bovine cell activity). However, certain alpha-1/alpha-2 hybrid interferons had higher activities on mouse cells (100-170% of bovine cell activity). See also Weck, et al., Infection & Immun., 35:660 (1982), Weck, et al., J. Gen. Virol., 64:415-419 (1983).
Bell, EP Appl 163,993 (publ. Dec. 11, 1985) ,Bell, EP Appl 131,818 (publ. Jan. 23, 1985), and Bell, EP Appl 130,564-566 (publ. Jan 9, 1985), describe the preparation of a beta-IFN analogue by rDNA techniques and suggests that it may be useful in treating viral infection ('993, claim 36). No reference is made to the creation of transgenic animals.
Itoh, EP Appl 83,069 and Crea, EP Appl 48,970 disclose bacterial expression of a human beta-IFN gene under the control of a trp promoter. Guarente, EP Appl 42,246 also relates to bacterial expression of beta-IFN, but as part of a hybrid protein.
Mark, U.S. Pat. No. 4,588,585 relates to an IFN-beta analogue. It is suggested that this protein may be useful in diagnosis and treatment of viral and other infections. (col. 20)
Eppstein, U.S. Pat. No. 4,606,917 discloses a synergistic composition of DHPG and beta-IFN for use in treating antiviral infections.
Thompson, EP Appl 217,645 describes a stabilized IFN-beta or IL-2 composition for antiviral use.
TORAY, JP 61052285 (abstract) discloses expression of IFN-beta in a eukaryotic cell, under control of, for example, the MT promoter.
Gruneberg, EP Appl 34,306 (abstract) describes expression of IFN-beta in a microorganism.
Sugano, WO 82/02715 (abstract) describes expression of IFN-beta in a eukaryotic cell.
Palmiter, W083/01783 teaches that a structural gene may be coupled to the MT promoter, introduced into the embryo of a mouse, and expressed in the adult mouse after induction with heavy metals. Palmiter worked with both thymidine kinase and rat growth hormone. Palmiter suggested that the MT promoter could be used to control transcription of other genes in higher animals, including genes relating in some manner to "disease resistance". (page 31).
Hamer, W084/02534 discloses expression of HGH in mammalian cell culture under control of the MT promoter.
Karin, U.S. Pat. No. 4,601,978 generally suggests inducible expression of heterologous genes in mammalian cells under the control of the human MT II gene.
Kushner, W086/04920 relates to expression of HGH and certain other proteins (IFN is not mentioned) under the control of the human MT II promoter in CHO cells.
The foregoing information is disclosed because it might be considered material by the Examiner in the course of examining this application. No admission is made that any of this information constitutes prior art or pertinent prior art, that the references themselves accurately or completely describe the work reported, or that the dates of publication given are exact.